Cd27 pe cy7

Manufactured by BioLegend

Sourced in United States

CD27-PE-Cy7 is a fluorochrome-conjugated monoclonal antibody that binds to the CD27 cell surface antigen. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on various lymphocyte subsets. This product is designed for use in flow cytometry applications.

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Lab products found in correlation

Facsaria 3, by BD (3 mentions) Cd19 bv510, by BioLegend (2 mentions) Cd3 bv650, by BD (2 mentions) Zombie uv live dead dye, by BioLegend (2 mentions) Igd fitc, by BioLegend (2 mentions) Cd19 apc, by BioLegend (2 mentions) Cd3 apc h7, by BD (1 mentions) Percp cy5.5 cd45ra, by BioLegend (1 mentions) Bv605 ccr6, by BioLegend (1 mentions) Cd4 alexa fluor 700, by BioLegend (1 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Cd45ra apc h7, by BD (1 mentions) Cd14 bv510, by BioLegend (1 mentions) Cd4 pe, by BD (1 mentions) Cell fix solution, by BD (1 mentions) Percp cy5.5 cd8, by BD (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions) Cd71 pe, by BioLegend (1 mentions) Spectroflo v2, by Cytek (1 mentions) Cd19 bv750, by BioLegend (1 mentions)

14 protocols using cd27 pe cy7

Multiparameter Flow Cytometry Analysis of T Cell Subsets

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Flow cytometry was used to measure the expression of cell markers (LSR II, BD Biosciences). Quantification of α4β7 levels was performed by gating β7⁺CD45RA⁻ within the population of CD4⁺ T cells to identify α4β7^hi populations based on previously published data that demonstrated that αEβ7, the only other form of β7 found on human T cells, is rarely found in the blood [2 (link),13 16 (link),17 ]. PBMCs were stained with the following combination of antibodies from BD Biosciences: APC-H7-CD3 (560176), PerCP-Cy5.5-CD45RA (563429), BV421-CCR5 (562576), PE-Integrin β7 (555945), BB515-CD25 (564467), PE-Cy7-CD27 (560609), PE-CF594-HLA-DR (562304), BUV395-CD8 (563795), BUV496-CD38 (564657); and the following antibodies from Biolegend: BV605-CCR6 (353420), AlexaFluor 700-CD4 (344622), BV510-CCR7 (353232), AlexaFluor 647-CD127 (351318) (Supplemental Figure S1).

Martin A.R., Patel E.U., Kirby C., Astemborski J., Kirk G.D., Mehta S.H., Marshall K., Janes H., Clayton A., Corey L., Hammer S.M., Sobieszczyk M.E., Arthos J., Cicala C., Redd A.D, & Quinn T.C. (2020). The association of α4β7 expression with HIV acquisition and disease progression in people who inject drugs and men who have sex with men: Case control studies. EBioMedicine, 62, 103102.

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Comprehensive Immune Phenotyping of PBMCs

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PBMCs were initially stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFischer Scientific, Waltham, MA, USA) for 20 min before surface staining with conjugated antibodies in washing buffer for 20 min and fixed with CellFix solution (BD Biosciences, San Jose, CA, USA). Commercial conjugated antibodies used included: BV785-CD3, BV510-CD14, BV510-CD16, BV510-CD19, PE/Dazzle594-CD39, BV711-CD103, BV421-CCR7, PE/Cy7-CD27 (BioLegend Inc, San Diego, CA, USA); PE-CD4, PerCP/Cy5.5-CD8, BV650-PD1, BB515-Tim3, APC/H7-CD45RA (BD Bioscience, San Jose, CA, USA). All samples were acquired by a four-laser BD LSRFortessa flow cytometer (BD Company, Franklin Lakes, NJ, USA) and analyzed by FlowJo software v.10.6 (FlowJo Co, San Diego, CA, USA) [Figure 1].

Dong D.N., Fan P.W., Feng Y.N., Liu G.H., Peng Y.C., Dong T., Wang R.Z, & Yu J.M. (2021). Association between circulating CD39+CD8+ T cells pre-chemoradiotherapy and prognosis in patients with nasopharyngeal carcinoma. Chinese Medical Journal, 134(17), 2066-2072.

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Multiparametric Flow Cytometry Staining

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Staining for flow cytometry analysis was performed using cryopreserved PBMCs. Cells were stained for 30 min on ice with biotinylated recombinant HAs diluted in in 2% FBS and 2 mM EDTA in PBS (P2), washed twice, then stained for 30 min on ice with Alexa 647-conjugated S, IgA-FITC (M24A, Millipore, 1:500), IgG-BV480 (goat polyclonal, Jackson ImmunoResearch, 1:100), IgD-SB702 (IA6–2, Thermo, 1:50), CD38-BB700 (HIT2, BD Horizon, 1:500), CD20-Pacific Blue (2H7, 1:400), CD4-BV570 (OKT4, 1:50), CD24-BV605 (ML5, 1:100), streptavidin-BV650, CD19-BV750 (HIB19, 1:100), CD71-PE (CY1G4, 1:400), CXCR5-PE-Dazzle 594 (J252D4, 1:50), CD27-PE-Cy7 (O323, 1:200), IgM-APC-Fire750 (MHM-88, 1:100), CD3-APC-Fire810 (SK7, 1:500), and Zombie NIR (all BioLegend) diluted in Brilliant Staining buffer (BD Horizon). Cells were washed twice with P2 and acquired on an Aurora using SpectroFlo v2.2 (Cytek). Flow cytometry data were analyzed using FlowJo v10 (Treestar). In each experiment, PBMC were included from convalescent and control participants.

Turner J.S., Kim W., Kalaidina E., Goss C.W., Rauseo A.M., Schmitz A.J., Hansen L., Haile A., Klebert M.K., Pusic I., O’Halloran J.A., Presti R.M, & Ellebedy A.H. (2020). SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans. Research Square.

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Multiparameter Flow Cytometry of CSF Immune Cells

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Flow cytometry was conducted using an LSRFortessa (BD Biosciences). A panel consisting of antibodies conjugated to six different fluorophores was used to classify subsets of memory T cells and for drop-seq. Antibodies used were: CD8α-Pacific blue (BioLegend), CD3-BV650 (BD Biosciences), CD45RA-APC-Cy7 (BioLegend), CCR7–488 (Bio-Legend), IL-7Rα-PE (BioLegend) and CD27-PE-Cy7 (BioLegend). For characterization of CSF cells, this same panel was used, but CD19-PE-Cy5 (BioLegend) and CD14-Qdot-705 (Thermo Fisher Scientific) were included. For sorting CSF T cells for TCR plate-seq, the following antibodies were used: CD8α-PE (BioLegend), CD161-PE-Cy7 (BioLegend), CXCR3-APC (BioLegend), CD4-APC-Alexa700 (Thermo Fisher Scientific), CD39-APC-Cy7 (BioLegend), CD38-FITC (BioLegend), PD-1-BV421 (BD Biosciences), CD45RA-BV605 (BD Biosciences), CD3-BV650 (BD Biosciences), CD27-BV786 (BD Biosciences) and CD127-BUV395 (BD Biosciences). For each experiment, a compensation matrix was developed using singly stained and unstained controls or fluorescent beads, and all analysis was conducted in Cytobank.

Gate D., Saligrama N., Leventhal O., Yang A.C., Unger M.S., Middeldorp J., Chen K., Lehallier B., Channappa D., De Los Santos M.B., McBride A., Pluvinage J., Elahi F., Tam G.K., Kim Y., Greicius M., Wagner A.D., Aigner L., Galasko D.R., Davis M.M, & Wyss-Coray T. (2020). Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer’s disease. Nature, 577(7790), 399-404.

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Phenotypic Analysis of Cryopreserved PBMCs

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Cryopreserved PBMCs were thawed and washed twice with 10 mL of FACS buffer (1 x PBS containing 2% FBS and 1 mM EDTA) and resuspended in 100 uL of 1x PBS containing Zombie UV live/dead dye at 1:200 dilution (BioLegend, 423108) and incubate at room temperature for 15 minutes. Following washing, cells were incubated with an antibody cocktail for 1 hour protected from light on ice. The following antibodies were used: IgD PE (BD Biosciences, 555779), IgM PerCP-Cy5.5 (BioLegend, 314512), CD20 APC-H7 (BD Biosciences, 560734), CD27 PE-Cy7 (BioLegend, 302838), CD14 PE/Dazzle^™ 594 (BioLegend, 301852), CD16 BV605 (BioLegend, 302040), IgG BV650 (BD Biosciences, 740596), CD3 BUV737 (BD Biosciences, 612750) and Alexa Fluor 488-labeled Wuhan spike (SinoBiological, 40589-V27B-B), and BV421 labeled Omicron Spike (SinoBiological^™, 40589-V49H3-B). All antibodies were used as the manufacturer’s instruction and the final concentration of each probe was 0.1 ug/ml. Cells were washed twice in FACS buffer and immediately acquired on a BD FACS Aria III for acquisition and FlowJo for analysis.

Wimmers F., Burrell A.R., Feng Y., Zheng H., Arunachalam P.S., Hu M., Spranger S., Nyhoff L., Joshi D., Trisal M., Awasthi M., Bellusci L., Ashraf U., Kowli S., Konvinse K.C., Yang E., Blanco M., Pellegrini K., Tharp G., Hagan T., Chinthrajah R.S., Grifoni A., Sette A., Nadeau K.C., Haslam D.B., Bosinger S.E., Wrammert J., Maecker H.T., Utz P.J., Wang T.T., Khurana S., Khatri P., Staat M.A, & Pulendran B. (2023). Systems biological assessment of the temporal dynamics of immunity to a viral infection in the first weeks and months of life. medRxiv.

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Multiparametric Flow Cytometry Analysis

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Single cell suspensions from tissue and PBMCs were analyzed on a Gallios 10-color flow cytometer (Beckman Coulter) on the day of sample acquisition. At least 5 × 10⁵ events per sample were acquired and lymphocyte subsets were determined using anti-human antibody-conjugates CD45-PE-eFluor610 (HI30; eBioscience), IgD-FITC (clone: IA6-2), CD86-PE (IT2.2), CD86-BV421 (IT2.2), CD38-PerCP/Cy5.5 (HIT2), CD27-PE/Cy7 (O323), CD21-APC (Bu32), CD138-Alexa Fluor 700 (MI15), CD19-APC/Cy7 (HIB19), CD19-APC-Fire 750 (SJ25C1), CD20-Pacific Blue (2H7), CD24-FITC (ML5), CD25-Alexa Fluor 700 (BC96), CCR7-FITC (G043H7), ICOS-PE (C398.4A), CD4-PerCP/Cy5.5 (RPA-T4), CD8-PE/Cy7 (HIT8a), PD1-APC (EH12.2H7), CD45RA-Alexa Fluor 700 (HI100), CD3-APC-Cy7 (HIT3a), CD3-Alexa Fluor 700 (SK7), Interleukin-10-PE (JES3-19F1), CXCR5-PB (J252D4; all Biolegend) and aqua dead cell stain (Life Technologies). Intracellular IL-10 staining was performed using an intracellular staining kit purchased from Biolegend. After live/dead and surface staining cells were fixed and permeabilized according to the manufactures protocol.

Lechner A., Schlößer H.A., Thelen M., Wennhold K., Rothschild S.I., Gilles R., Quaas A., Siefer O.G., Huebbers C.U., Cukuroglu E., Göke J., Hillmer A., Gathof B., Meyer M.F., Klussmann J.P., Shimabukuro-Vornhagen A., Theurich S., Beutner D, & von Bergwelt-Baildon M. (2019). Tumor-associated B cells and humoral immune response in head and neck squamous cell carcinoma. Oncoimmunology, 8(3), 1535293.

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Phenotyping of B cell subsets

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For phenotyping of B cells, CD9⁺ and CD9⁻ B cells, the antibodies used were as follows: CD27-PE-Cy7 (clone 0323, Biolegend), CD38-PE (clone HB7, Biolegend), CD19-BV510 (clone HIB19, Biolegend), CD24-APC (clone SN3 A5-2H1D, eBioscience, San Diego, CA, USA), HLA-DR-PERCP (Clone L243, Biolegend), CD40-PE (Clone 5C3, BD Biosciences), CD86-PE (Clone 2331, BD Biosciences), CD25-PE/Cy5 (Clone M-A251, BD Biosciences), CD69-APC (Clone FN50, Biolegend), IgD-APC-Cy7 (Clone, IA6-2, Biolegend), IgM-FITC (Clone, SA-DA4, eBioscience), CD5-FITC (Clone, L17F12, eBioscience), CD10-APC (Clone, LT10, ImmunoTools, Friesoythe, Germany), CD20-PE-Dy647 (Clone, LT20, ImmunoTools) and CD21-FITC (Clone, LT21, ImmunoTools). Viability was assessed by 7AAD staining (BD Biosciences). All flow cytometric acquisition was performed with FACS Canto II, BD Biosciences. The data analysis was performed using FlowJo V7 (TreeStar Inc., Ashland, OR, USA). The gating strategy for the Bregs and single markers can be found in Supplementary Figure S1.

Mohd Jaya F.N., Garcia S.G., Borras F.E., Guerrero D., Chan G.C, & Franquesa M. (2021). In Vitro Characterization of Human CD24hiCD38hi Regulatory B Cells Shows CD9 Is Not a Stable Breg Cell Marker. International Journal of Molecular Sciences, 22(9), 4583.

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Murine Liver Immune Cell Isolation

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Murine mononuclear cells from livers were isolated as previously described (8 (link), 15 (link)). Erythrocytes were lysed using RBC lysis buffer (BioLegend, San Diego, CA). Cells isolated from livers were treated with Fc Block antibody (anti CD16/32, BD Biosciences). Cells were stained with mouse-specific immune cell surface markers for 30 min at 4°C. The following anti-mouse antibodies were used at a dilution of 1:200: CD3-APC, CD3-PerCP-Cy5.5, NK1.1-APC, NK1.1-PE, CD19-FITC, CD69-FITC, CD27-PE-Cy7, CD11b-PE, CD11b-PErCP-Cy5.5, Gr1 V450 and IFN-γ-FITC (Biolegend, San Diego, CA). For staining of IFN-γ, cells were treated with Cytofix/Cytoperm (BD) following initial cell-surface staining and then performed intracellular staining.

Teng K.Y., Han J., Zhang X., Hsu S.H., He S., Wani N., Barajas J., Snyder L.A., Frankel W.L., Caligiuri M.A., Jacob S.T., Yu J, & Ghoshal K. (2016). Blocking the CCL2-CCR2 axis using CCL2 neutralizing antibody is an effective therapy for hepatocellular cancer in a mouse model. Molecular cancer therapeutics, 16(2), 312-322.

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SARS-CoV-2 Protein-Specific B-Cell Sorting

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For protein-specific B-cell sorting, cryopreserved B cells from five convalescent patients (rAbCOVID29, rAbCOVID27, rAbCOVID09, rAbCOVID07, and rAbCOVID19) were selected due to the availability of cells from these patients for this experiment (Figure S1). The cells were thawed, washed with Cell Staining Buffer (BioLegend, San Diego, CA, USA), and then incubated at 4°C for 20 min with a cocktail of antibodies and protein S1 and RBD (WT) tetramers. The cocktail consisted of CD19-BV421 (BioLegend clone HIB19), CD27-PeCy7 (BioLegend clone O323), IgD-FITC (BioLegend clone IA6-2), RBD-APC, and S1-PE tetramers. After incubation, the cells were washed twice, and then the stained cells from each patient were pooled. Finally, protein-specific B cells were gated as CD19⁺CD27⁺IgD^-RBD⁺S1⁺, CD19⁺CD27⁺IgD^-RBD⁺S1^- and CD19⁺CD27⁺IgD^-S1⁺RBD^- and sorted using a BD FACS Aria III (BD Biosciences, San Jose, CA, USA).

García-Vega M., Melgoza-González E.A., Hernández-Valenzuela S., Hinojosa-Trujillo D., Reséndiz-Sandoval M., Llamas-Covarrubias M.A., Loza-López M., Valenzuela O., Soto-Gaxiola A., Hernández-Oñate M.A., Mata-Haro V., Cassaniti I., Sammartino J.C., Ferrari A., Simonelli L., Pedotti M., Sun R., Zuo F., Baldanti F., Varani L., Marcotte H., Pan-Hammarström Q, & Hernández J. (2023). 19n01, a broadly neutralizing antibody against omicron BA.1, BA.2, BA.4/5, and other SARS-CoV-2 variants of concern. iScience, 26(4), 106562.

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Flow Cytometry Analysis of Immune Cell Profiles

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Flow cytometric analysis of PBMCs was performed pre-transplant, weekly thereafter and at terminal endpoint on all recipients. In addition, cells were isolated from lymph nodes and spleen tissue recovered at euthanasia and subjected to similar flow analysis. Cells were stained with the following fluorophore-conjugated antibodies: CD19-PE (Beckman Coulter, Indianapolis, IN, USA), CD28-PE-Cy7, CD10-PerCP-Cy5.5, CD95-eFlour450, CD27-PE-Cy7 (Biolegend, San Diego, CA, USA), CD25-PE (Miltinyi Biotech, Bergisch Gladbach, Germany), IgD-FITC (Southern Biotech, Birmingham, AL, USA), CD2-PE, CD3-APC-Cy7(-Pacific Blue, or PerCP), CD4-PerCP-Cy5.5, CD8-BV510, CD20-APC-Cy7, CD45-PE-Cy7, CD69-FITC, IgM-BV510, and Ki67-FITC (BD Bioscience, San Jose, CA, USA). Secondary analysis of data was performed using FlowJo software (Tree Star, San Carlos, CA, USA).

Gao Q., Davis R., Fitch Z., Mulvihill M., Ezekian B., Schroder P., Schmitz R., Song M., Leopardi F., Ribeiro M., Miller A., Moris D., Shaw B., Samy K., Reimann K., Williams K., Collins B, & Kirk A. (2021). Anti-thymoglobulin Induction Improves Neonatal Porcine Xenoislet Engraftment and Survival. Xenotransplantation, 28(6), e12713.

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